Synaptic dysfunction is the basis of disorders of affect, learning, memory and motor control. The long-term goal of the proposed research is to understand the regulation of transmitter release at central synapses, which are the fundamental elements of neuronal communication and provide the basis for many mechanisms underlying adaptive properties of neuronal networks. Functional correlates of the quantal model of exocytosis, which allows identification of loci of synaptic plasticity remain controversial, in particular, the determinants of the stochastic nature of release. The first aim is designed to test the range of validity of a specific hypothesis that correlates structure and function at single synaptic units, namely the hypothesis that an action potential triggers the release of at most one vesicle per presynaptic active zone. Accordingly, the number of quantal units determined by statistical analysis of response fluctuations should not exceed the number of active zones comprising a synaptic connection. Statistical analyses of evoked responses will be combined with ultrastructural data on synaptic connectivity in two preparations: I) identified excitatory connections in goldfish between the presynaptic Mauthner (M-) axon and the postsynaptic axon of cranial relay neurons (CRNs), and ii) single inhibitory eynapses on somata of cultured rat medullary and spinal cord neurons. Extracellular Ca++ will be varied, to allow different baseline levels of release. The second aim is to test the hypothesis that the spontaneous or miniature quantal unit is not necessarily the building block of the stimulus evoked response but does provide insight concerning the vesicle cycle and the kinetics of release. Specific issues are whether quantal size is smaller than minis during synaptic depression at the M-axon to CRN connection and whether evoked responses have skewed decay time distributions comparable to those of minis. Alternatively, do slow minis signify a slower release of single vesicles than occurs after an action potential? The last aim is to test the hypothesis that depression at the M-axon to CRN connection is not due to a simple depletion of the available vesicle pool but that instead a presynaptic Ca++ influx triggers two competing processes, synchronous release and transient depression. For this purpose, quantal analysis of frequency dependent and paired pulse depression will be combined with manipulations of release, such as presynaptic injections of Ca++, specific kinases, antibodies and peptides. The results form all aims will allow a more unified and coherent view of the molecular mechanisms of transmitter release and its uncertainty.